Guanine nucleotide dissociation inhibitor (GDI) is a critical component of the synapse and likely to regulate the recycling of the small GTPases belonging to the Rab3 subfamily. Rab3A is believed to regulate a key step in synaptic vesicle targeting and/or fusion to the presynaptic membrane. We have generated crystals of GDI which diffract to 1.8 A and have obtained a preliminary structure using X-ray crystallography at 2.9 A resolution. A major goal of this project will be the determination of the three- dimensional structure of GDI to elucidate the molecular basis for the interaction of GDI with Rab3A during neurotransmission. We will generate site-directed mutants in GDI and Rab3A to characterize amino acid residues involved in complex formation. In the case of Rab3A, these studies will include mutants ina the guanine nucleotide binding motifs, the effector domains, and the N- and C-hypervariable domains. In the case of GDI, these will involve selective substitutions, deletions and truncations based on the structure of GDI as derived from X-ray crystallography. To establish the functional properties of such mutants, we will use a range of biochemical assays which provide sensitive measures for GDI-Rab3A complex formation, guanine nucleotide dissociation inhibitor activity and the capacity of GDI to extract and deliver Rab3A to biological membranes. The goal of these experiments will be to define a molecular and biochemical basis for GDI specificity and function in recognition of Rab proteins. Using results obtained from these studies, we propose to analyze the physiological role of GDI in vitro utilizing permeabilized synaptosome preparations. We will measure the functional sensitivity of transmitter release and the recycling of Rab3A to recombinant forms of GDI which many function as trans dominant inhibitors of Rab3A function. In general, structural knowledge of GDI and its Rab3A complex will provide a rigorous foundation to pursue the functional role GDI and Rab3A the synapse. Given the central role of GDI ifn the recycling of Rab3A proteins throughout the endocytic and exocytic pathway, knowledge of the structure of GDI and its Rab3A complex may provide important insight into presynaptic neurological disorders.